Fluidic force discrimination

ABSTRACT

This invention describes a method of using controlled fluidic forces to improve the performance of a biochemical binding assay where a target molecule is captured by specific molecular recognition onto a substrate surface with an affinity coating, and then labeled with a detectable micrometer-scale particle using a second specific molecular recognition reaction with the target. By using specific ranges of label sizes and laminar flow conditions, controlled fluidic forces can be applied to the label particles in order to selectively remove molecules bound to a surface according to their binding strength, and thereby increase the ratio of specifically bound labels to more weakly attached non-specifically bound labels. This method can be used with a wide variety of label types and associated detection methods, improving the sensitivity and selectivity of a broad range of binding assays.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention pertains to a method to selectively removemolecules bound to a surface according to their binding strength byattaching micrometer-scale particles to the bound molecules and thenapplying controlled, laminar fluidic forces to the particles. Such“fluidic force discrimination” (FFD) can be used to improve thesensitivity and selectivity of biochemical binding assays in many fieldsof use, including forensics, agriculture, medical diagnostics, food andwater safety, and anti-terrorism.

[0003] 2. Description of Related Art

[0004] Binding assays such as immunoassays, DNA hybridization assays,and receptor-based assays are widely used to detect trace quantities ofspecific target molecules contained in a complex sample. Typically, asolid substrate is coated with receptor molecules that have a specificbinding affinity for the target. When a liquid sample containing thetarget is applied to the substrate, the target biomolecules are capturedonto the surface by molecular recognition. This capture can beaccomplished via any specific ligand-receptor combination such asantibody-antigen or other specific binding combination such ascomplementary sequences of polynucleic acids (DNA, RNA, or PNA).

[0005] A common approach to detecting captured target molecules is tochemically attach to them a label that generates an observable signal.For example, a label can include a radioactive isotope, enzyme,fluorescent molecule(s) or magnetic particle. The attachment can be madevia any chemical means, but is usually made with very strong attachmentchemistry such as covalent or aminated electrostatic bonding, or viahigh-affinity molecular recognition to a second, exposed part of thecaptured target molecule. The label is detected by appropriate means asa measure of the concentration of the target in the sample. Detectionmethods have been developed based on a range of transduction mechanisms,including optical, electrical, magnetic, radioactive, electrochemical,thermal, and piezoelectrical. One example is illustrated in FIG. 1 for acase where a plurality of capture molecules specific to differenttargets are separately immobilized on a substrate with built-in sensorsfor the label particles. Note that for micrometer-scale labels thisillustration is not to scale: the target and receptor molecules aretypically 10 to 1000 times smaller than the labels.

[0006] There are many variants to binding assays using labels, but acommon goal is to measure the concentration of the target with as muchsensitivity and selectivity as possible. As long as a sufficient numberof labels are present to generate a detectable signal, the sensitivityand selectivity of a binding assay can be limited by labels bound to thesurface but not bound to a captured target (sometime referred to as“background” signal). Such labels may be attached to molecules that werealso present in the sample and have bound to the surface throughrelatively weak, non-specific bonds. Alternately, labels may be directlybound to the surface by buoyant weight and/or non-specific bonds. Theability to selectively remove labels bound non-specifically to thesurface can greatly improve the sensitivity and selectivity of a bindingassay by lowering the minimum number of labels that can be associatedwith confidence with the intended target, and reducing the likelihood ofwrongly associating detected labels with captured target, respectively.

[0007] It is possible to selectively remove labels boundnon-specifically to a surface in a binding assay by applying a force tothe labels sufficient to break weak, non-specific bonds but too small toremove those labels bound by the stronger bonds of specific molecularrecognition. On surfaces specially prepared to inhibit non-specificbinding, forces on the order of 1 pN are required for this purpose. Itis also possible to use the application of forces to label particles forthe purpose of selectively breaking specific bonds of increasingstrength and thereby either measure the rupture force or identify thebound target based on the rupture force. Forces>10 pN and as large as 1nN may be required for this purpose. U.S. Pat. Nos. 5,981,297 and6,180,418 describe the use of magnetically active beads and magneticforces to selectively remove non-specifically bound beads (BARC). U.S.Pat. Nos. 6,086,821 and 6,368,553 describe the use of ultrasonic energyto provide a variable force for measuring the binding forces betweenmolecular entities and for sensing the presence of an analyte in a testsample. The above referenced patents are incorporated by reference intheir entirety.

[0008] In many binding assays it is common practice to use some type ofrinsing step for the purpose of reducing the background signal. Commonrinsing methods include soaking with our without mechanical agitationand spraying with non-steady flow. Under these poorly controlledconditions smaller particles are removed with greater difficulty becauseof the no-slip boundary condition at the walls. For sufficiently largefluid boundary dimensions and flow velocities, fluid inertia leads toturbulent flow that can enhance particle removal. At small enough fluidboundary dimensions and velocities, however, the flow is determined byviscosity alone, leading to a steady, laminar flow that does not easilyremove particles from surfaces. The two regimes are characterized by theReynolds number, R_(e)=ρdv/η, where p is the fluid density, d is thecharacteristic dimensions of a channel (volume/surface area), v is thefluid velocity, and η is the viscosity. For particles or channels lessthan 1 mm wide and velocities less than 1 mm/s, R_(e) is less than 1 andthe flow is definitely laminar. The dividing line between laminar andthe beginning of turbulent flow is at R_(e)˜2000. Laminar flowconditions commonly occur in blood vessels and in the microfluidicssystems used in many biosensor systems.

[0009] Viscous hydrodynamic forces on particles at a wall have beenstudied with the goal of understanding cell adhesion. For example, themigration of white blood cells occurs (via molecular control) as a slowsticky-rolling detachment along blood vessel walls. Chang and Hammer,Langmuir, 1996, 12, 2271-2282 developed a lever amplification model andsimulated forces on molecules binding particles to a wall in fluid flowtangential to the binding surface. Zocchi observed the normal forcecomponents on 4.5 μm-diameter particles in a tangential flow(Biophysical J. 2001, 81, 2946-2953). He measured a lever amplificationof the flow forces and observed the rupture of biotin-streptavidin bondsand applied the results to the interpretation of cell adhesion assays.

SUMMARY OF THE INVENTION

[0010] This invention describes a simple, versatile method ofcontrollably removing non-specifically bound micrometer-scale particlelabels in a binding assay using fluidic flow drag forces, which we willcall fluidic force discrimination (FFD). FFD can only be achieved usingspecific ranges of label sizes and flow conditions. FFD can be used witha wide variety of label types and associated detection methods,improving the sensitivity and selectivity of a broad range ofsolid-phase binding assays. FFD could also be potentially used to applycontrolled, variable forces to identify or separate different targetsbased on the force required to rupture bonds between a plurality ofparticle-labeled targets and the substrate-bound receptors.

FIGURES

[0011]FIG. 1. depicts capture molecules specific to different targetsseparately immobilized on a substrate with built in sensors for thelabel particles.

[0012]FIG. 2. depicts the lever action and resultant tension, T, in amolecular tether.

[0013]FIG. 3. depicts the action of FFD on non-specifically bound beadswhich influences remaining beads and signal to background ratios forbeads used in the assay of the invention.

[0014]FIG. 4. depicts the assay described in the experimentaldemonstration of this invention. Note that the figure is not to scale.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

[0015] Biomolecular recognition has been widely discussed in thescientific literature. The terms ligand and receptor are most often usedwith protein recognition such as antigen-antibody, but in the inventiondescribed herein, we shall take a broader definition that also includesbut is not limited to specific molecular recognition of enzymes andsubstrates, chelators and ions, and complementary strands of polynucleicacids DNA, RNA, and PNA.

2. Theory and Advantages

[0016] The present invention uses tangential laminar fluidic forces onparticles or beads bound to a surface to selectively break the bondsbinding the beads. In particular, FFD can remove a background of moreweakly attached, non-specifically bound beads. Under laminar flow, thefluid velocity and associated viscous fluidic force on a particle isgreatly reduced at a surface. Solving the Navier-Stokes equation forlaminar flow in a tube, for example, leads to parabolic variation ofaxial fluid velocity across the fluid channel. But near the wall, thevelocity increases linearly from zero with distance z away from thewall. Dropping higher order terms, the velocity is given by v=4 uz/R forz<<R, where u is the mean velocity in the channel, z is the distancefrom the wall, and R is the tube radius. The force on a stationaryparticle in bulk fluid flow is given by the Stokes drag F_(s)=6πηav_(c),where a is the particle radius and vC is the fluid velocity at theparticle center. Similarly the torque in bulk fluid with uniform shearis Γ_(s)=4πηa²V_(c). Exact laminar solutions for a stationary particleat a wall in a semi-infinite fluid give F_(e)=1.7005*6πηav_(c) andΓ_(e)=0.94399*4πηa²v_(c), Goldman et al. Chem. Engr. Sci., 1967, 22653-660.

[0017] For particles bound to a surface by a flexible molecular“tether,” there can be a lever amplification of this Stokes force andassociated torque acting on the particle. The lever action and resultanttension, T, in the molecular tether is illustrated in FIG. 2. Here thetether bends or pivots and the particle then makes sliding contactdownstream at a height difference of h above the tether/wall attachmentpoint. The height h varies with the surface roughness of the wall andbead. For a bead of radius, a, in the micrometer range and abiomolecular tether of much smaller length, L˜10 nm, the angle betweenthe tether attachment and bead contact, θ, is small and the angle of thetether, φ, is close to 90 degrees. In this case, the tangential forcebalance is F=T cos φ and the normal force balance is N=T sin φ, where Nis the force the surface exerts on the bead. The torque balance aboutthe particle center yields Γ=−aT cos(φ+θ). With the geometricalcondition (L-h)sin φ=aθ²/2, small θ, and φ almost 90 degrees, thetension on the molecular tether is$T \cong {\left( {F_{e} + \frac{\Gamma_{e}}{a}} \right){\sqrt{\frac{a}{2\left( {L - h} \right)}}.}}$

[0018] For a 1.4 μm-radius bead and a 10 nm tether, the tension createsa lever amplification of the purely tangential fluidic force of aboutten times. Overall, given the linear velocity gradient near surfaces,the Stokes linear particle size dependence, and the lever action, thetension is proportional to a^(2.5). For a smooth, 1.4 μm-radius bead ata wall where the velocity at the bead center is 100 μm/s, the exactStokes force is 4.5 pN and the total amplified tension (including thetorque from the 10 nm tether) is 51 pN. This force is over 100 timeslarger than the normal force ˜0.3 pN exerted by a large, high magneticfield NdFeB magnet on a commercial paramagnetic particle of the samesize, Edelstein et al., Biosensors and Bioelectronics, 2000, 14,805-813. Note that multiples of the single tether rupture force may berequired to remove beads bound with more than one tether, although thedensity of binding sites on the bead and/or substrate surfaces can beadjusted to greatly reduce the probability of multiple bead-surfacebonds.

[0019] The application of force to biomolecular bonds, and in generalchemical bonds, decreases the energy barrier to dissociation. Therefore,the thermally-activated dissociation rate of chemical bondsexponentially decreases with binding energy but exponentially increaseswith the application of an external force, as originally discussed byBell, Science, 1978, 200, 618-627. The dissociation rate k is given by${k = {v_{0}^{- \frac{E_{b}}{kT}}^{\frac{Fx}{kT}}}},$

[0020] where v₀ is the attempt frequency, E_(b) is the binding energy, Fis the force or tension T on the bond, x is the bond length extensionfor the transition-state at the top of the dissociation barrier(referred to as the “barrier length” and typically 0.1 to 1.5 nm), andkT is the thermal energy (4.1 pN·nm at 25° C.). Thus, the dissociationrate can be increased ˜100 times with the application of a ˜100 pN force(assuming x˜0.2 nm). Such effects have been observed experimentallyusing forces applied mechanically with an atomic force microscope, Leeet al., Langmuir, 1994, 10, 354-357; Merkel et al., Nature, 1999, 397,50-53; Evans, Annu. Rev. Biophyis. Biomol. Struct. 2001, 30, 105-128.

[0021] Laminar fluidic force removal of particles enables much moreuniform and controlled fluidic forces then conventional rinsing methods.Significantly, the forces can be controlled by choice of specificparticle size and flow conditions (flow rate and channel geometry). Forparticle-labeled binding assays, using FFD to selectively removenon-specifically bound particles and thereby enable a lower number ofspecifically bound labels to be detected with confidence has manyadvantages over other discrimination methods. In particular, becausemany of the most sensitive assay approaches already include a fluidicsystem, FFD can be easily implemented by proper design of the fluidics,therefore removing the requirement for external rinsing, reducing theassay time and simplifying the assay protocol. A significant advantageof FFD is that it can be used with any type of particle label ofappropriate size and functionality, including but not limited tofluorescent, luminescent, light-scattering, magnetic, or radioactive.

[0022] Compared to magnetic force discrimination methods, FFD does notrequire paramagnetic beads or an external magnet field source. Inaddition, currently available commercial paramagnetic particles can onlygenerate forces in the ˜1 pN range, insufficient for forcediscrimination between specific ligand-receptor bonds. Compared toultrasonic force discrimination, FFD does not require a separateapparatus for generating ultrasonic energy, and avoids potentialproblems associated with internal flow cell acoustic reflections causinga variation in the magnitude of local ultrasonic forces as a function ofposition along a wall.

3. Experimental Demonstration—DNA Hybridization Assay with MicrobeadLabels

[0023] The initial observation of FFD was made using Dynal M280microbead labels (Dynal, Oslo, Norway). The substrate was a microsensorchip fabricated on a silicon wafer and then covered with a 40 nm-thickgold film. The gold coating was required for covalent attachment of thebiomolecules using thiol bonds (S-Au). After coating with gold, thechips were stored in a dry Nitrogen chamber. The chip surfaces werecleaned before use by rinsing with ethanol and distilled water.

[0024] Spots of single-stranded DNA (ssDNA) oligonucleotide receptors(10 μM in 400mM potassium phosphate, pH 7.0) 250 μm in diameter weredeposited onto the surface using Rapidograph® pen tips, Sheehan et al.Biosensors and Bioelectronics, 2003, 18, 000. Two alkanethiol-terminatedssDNA capture receptors were used for the experiment. The positivecapture receptor, a 25-base-long ssDNA sequence complementary to thessDNA target, was terminated on the 3′ end with six additional adeninenucleotides and three alkane spacers followed by the thiol. The negativecapture receptor was a 24-base-long ssDNA sequence non-complementary tothe target with 3 alkane spacers and a thiol on the 3′ end. Afterspotting, the chip was left for eight hours in a humid chamber at 37° C.to allow the ssDNA to immobilize onto the Au surface. (It is unclearwhether the length of time in the humid chamber is critical.)

[0025] A flow cell made of PDMS (polydimethylsiloxane), a transparentelastomer, was attached to the chip surface with double-sided adhesivetape so that fluid could be flowed through the cell directly in contactwith the chip surface. The flow cell had a cross-section of 100 μm highby 2.89 mm wide perpendicular to the flow and a length of 4.4 mm in thedirection of the flow. The chip and flow cell were mounted on an uprightmicroscope with sufficient magnification to directly observe thepresence and flow of individual beads within the flow cell and on thesurface. The microscope was equipped with a video camera connected to acomputer for digital image recording. The reagents were pumped throughthe flow cell at controlled rates with a peristaltic roller pump.

[0026] Thiolated PEG (5000 MW polyethylene glycol, Shearwater,Huntsville, Ala.) was introduced over the surface in the flow cell at aconcentration of 10 mg per mL deionized (DI) water and let stand for 1hour. The PEG formed a non-fouling region between the capture receptorspots of DNA. The application of PEG was not necessary for the assay butreduced the non-specific binding of target ssDNA and bead labels outsideof the intended capture spots, thereby increasing the number of targetssDNA and beads available to bind within the capture spots.

[0027] 2×SSC buffer (0.3 M NaCl, 30 mM sodium citrate, pH 7.0) with0.25% sodium dodecyl sulfate (SDS) was introduced and allowed to standfor 30 minutes. The salt and detergent buffers used here and insubsequent steps enhanced hybridization efficiency and reducedhydrophobic aggregation.

[0028] Target ssDNA (100 pM in 2×SSC with 0.25% SDS), complementary tothe positive capture receptor ssDNA, was flowed over the surface at arate of 2 μL/min for 15 minutes to allow for hybridization. The targetssDNA molecules were biotinylated so that they could be directly labeledwith streptavidin-coated microbeads (one microbead/DNA) after begincaptured via hybridization. The ssDNA target was composed of a25-base-long sequence complementary to the capture receptor terminatedon the 3′ end by six adenine nucleotides and a biotin molecule. Ahybridized target-capture DNA molecule was about 12 nm long from theAu-S bond to the biotin.

[0029] Streptavidin coated Dynal M280 microbead labels, 2.8 μm indiameter, were introduced from an aliquot with concentration of greaterthan or equal to 7×10⁶ beads/cm³ in 1×SSC with 0.125% SDS. Start/stopcycles of 10 seconds of flow at a flow rate of 10 μL/min followed by 50seconds at zero flow rate to allow the beads to settle depositedapproximately 200 beads/200 μm diameter circular spot per cycle. Afterfifteen 1 min cycles, approximately 2000 beads were within a 200 μmdiameter capture spot.

[0030] FFD was then used to selectively remove the more weakly attached,non-specifically bound beads as plotted in FIG. 3. 1×SSC buffer with0.125% SDS was flowed through the cell for 3 min at each of thefollowing flow rates: 10, 40, 60, and 110 μL/min. After each 3 minperiod the flow was stopped and the number of beads was counted withinthe 200 μm diameter spots containing the positive and negative capturereceptors and within an equivalent area on the PEG coated gold betweenthe capture spots. For the flow cell cross-section used and assuming 10nm tethers and smooth surfaces, these flow rates generate respectivefluid velocities at the center of each microbead of 48, 193, 290, and532 μm/s; shears of 34, 138, 207, 380 s⁻¹; calculated Stokes forcesF_(e) of 2.1, 8.6, 13, and 24 pN; and tether tensions (including torque)of approximately 25, 99, 149, and 273 pN. As shown in FIG. 3, therelative the number of beads specifically bound on a positive capturespot compared to the number bound non-specifically on either a negativespot or on the surrounding PEG-coated surface increases as the flow rateis increased to 60 μl/min. The increase is caused by the relativelygreater decrease in the number of beads bound to the negative spot andPEG-coated areas; i.e. those bound by non-specific interactions. Thedecrease in the number of beads in the positive spot can be attributedto a number of effects. First, there may be some beads non-specificallybound to ssDNA receptors that have not captured target ssDNA (beadswhose removal is desirable), which are expected to come off at lowerflow rates/forces. As the flow rate and forces increase, somebiotin-streptavidin bonds may also rupture. Under mechanical AFM forces,these bonds rupture at about 200 pN. Note that shear rupture of the muchstronger Watson-Crick base-pairing in the DNA is unlikely under theseconditions (the corresponding AFM rupture forces are >1 nN).

4. Preferred Embodiments

[0031] Given a large enough flow rate or bead diameter, or a shortenough molecular tether, FFD in principle can generate enough force tobreak any chemical bond. In most practical applications, FFD can be usedto identify or separate according to the required rupture forcepopulations of molecules bound to a surface by a plurality of differentbond strengths as long as the difference in the binding strengths islarger than variations in the force-times-barrier lengths. Forcevariations would include those caused by roughness or shadowing of flowacross one bead by a nearby upstream bead. For molecules withdissociation energetics that include multiple potential energy wells,such as many biological macromolecules, FFD using pulsed flow couldpotentially remove a relatively greater number of more weakly boundmicro-particle labels than strongly bound ones because of the relativelylonger times required to cross multiple energy barriers. FFD could alsobe used as a step in preparing a surface with a specific type or typesof molecules from a surface with an initial mixture of a plurality ofmolecules by selectively attaching microparticle labels to only thosetypes to be removed. After FFD, chemical methods (e.g., a change in pH)could be used to remove the label. Similarly, FFD could be used toadjust the density of molecules on a surface by randomly attachinglabels to some fraction, removing them by FFD, and then removing thelabels.

[0032] The following requirements for FFD focus on its use inbiomolecular binding assays, but similar requirements apply to a widerrange of potential applications.

[0033] FFD can be applied to a wide range of binding assays based onmolecular recognition. Primary examples are hybridization of targetssDNA with its complement, as in the experimental demonstrationdescribed herein, and antigen-antibody immunological assays. Toeliminate the requirement that a target be separately functionalized(e.g., with biotin), binding assays can be performed in a “sandwich”configuration. For example, for a hybridization assay, the capture ssDNAon the surface can be make shorter than the target so that the label canbe attached to the free end of the captured target through another ssDNAprobe complementary only to that free end. Similarly, in an immunoassaythe label can be attached through a second antibody specific to asecond, exposed region of the target antigen (a second epitope). Aperson skilled in the art of binding assays would know that there are amultitude of methods to attach a microparticle label to a target speciescaptured on a surface.

[0034] Any surface material can be used in FFD as long as it is suitablefor a binding assay. A person skilled in the art of binding assays wouldknow there are a multitude of substrates and corresponding chemicalmethods to attach useful capture molecules to said substrate surfaces.FFD will be produce the most uniform forces when the substrate surfaceis flat and smooth, with a height deviation, h, less than the tetherlength, L, over a spatial distance along the surface approximately equalto the bead radius. The smoothness reduces the variation in tension andremoval rates among the particle labels. For example, for 2.8μm-diameter beads and typical biomolecular tethers of length 10 nm, anh<2 nm over a 1.4 μm area of the surface will assure a <12% variation intension. Although not a requirement for FFD, this beneficial level ofsmoothness is not difficult to obtain for a person skilled in the art ofsubstrate preparation.

[0035] The requirements for FFD are well matched to the combination of˜1 μm scale beads commonly used (and commercially available) inbiomolecular binding assays and separations, and the ˜100 μm scalechannels typically used in microfluidics systems. The smallest bead sizeuseful for FFD is determined by the maximum flow velocities that are, inturn, constrained by either the maximum pressure tolerable in themicrofluidic system or, ultimately, by the maximum Reynolds number ofabout 2000 for controllable laminar (non-turbulent) flow. The pressureconstraint would typically limit the bead size for small height (<130μm) channels, with the creation of turbulence at high flow rateslimiting the bead size for larger channels. To quantify this, thevelocity at the height of the bead center, a, through a high-aspectratio rectangular flow cell like with height s, width w, length, l, andvolumetric flow, Q, is given for a<<h and w>>s to a good approximationby $v_{c} = {6{\frac{Q\quad a}{s^{2}w}.}}$

[0036] For pressure driven flow, Q can be written as${Q = \frac{s^{3}w\quad P}{12\eta \quad l}},$

[0037] where P is the pressure differential across the cell length andη, is the viscosity. From the above expressions for tension, the exactStokes force, and exact torque, the bead radius required to produce agiven tension on a tether of length L is given by$a^{2.5} = {\frac{T\quad l\sqrt{\left( {L - h} \right)}}{15.5\quad P\quad s}.}$

[0038] The equation for a tube of radius R with a<<R is the same exceptR replaces s.

[0039] The data in FIG. 3 show that the largest ratio of the positivelabel binding to the background label binding occurs in our experimentat a flow rate of 60 μL/min. For the 2.8 μm-diameter beads used, thisflow rate translates into a tension force on a 10 nm tether with h=0 ofabout 150 pN. Smaller beads would require a higher pressure, greaterflow cell height, or shorter tether to achieve the same tension force.For example, with our experimental flow-cell (with currently typicalmicrofluidic dimensions), increasing the pressure 5500 times to thepractical microfluidics pressure limit of 1 atmosphere would generatesimilar tension forces on 90 nm-diameter beads. Note, however, thatthese conditions would require an unpractical flow rate of 330 ml/min.

[0040] If higher pressures and flow rates were practical, for smallchannel heights the particle size limit is given by the requirement forlaminar flow,$a^{2.5} = {\frac{T\quad s^{2}\sqrt{\left( {L - h} \right)}}{7.45 \times 10^{5}\eta^{2}}.}$

[0041] For tension greater than or equal to 150 pN and the flow celldimensions in the experimental test case, this corresponds to arequirement for beads of diameter >66 nm. Note that nanometer-scalelabels are generally more difficult to detect then micrometer-scalelabels. Conversely, although beads>10 μm in diameter are generallyeasier to detect, the reduction in labels/surface area reduces thedynamic range in binding assays. In addition, larger beads require muchlower flow rates to achieve force discrimination, greatly increasing thetime required to introduce the labels (and the binding assay overall),which is an undesirable effect.

[0042] Although the examples presented in detail have used rectangularfluidic channels, this is not a requirement for FFD. The flow cell cantake a variety of enclosed forms, and flow could even be in an openchannel. The required fluidic conditions could also be achieved bymoving the substrate through a fixed fluid volume. Instead of tangentialflow across a substrate surface, FFD could also be achieved with fluidflow through and normal to a porous membrane on which beads were boundto the surface (although there would be no lever amplification of forceson the molecular tethers for this normal force).

1. A method for controllably removing non-specifically boundmicrometer-scale particle labels in binding assays, comprising: A.treating a capture surface to which a sample has been exposed in abinding assay with a detectable micrometer-scale particle label bondableto a molecule in said sample to be measured by said binding assay; B.employing a fluidic force discrimination system to removenon-specifically bound detectable labels, wherein said fluidic forcediscrimination system comprises a laminar flow system, said flow systemoperating for a time period and at a flow rate sufficient to establish ahigher ratio of specifically bound particles to non-specifically boundparticles, said micrometer-scale particle labels being detected by adetection means.
 2. The method according to claim 1, wherein saidfluidic force operates tangentially to the surface on which saidmicro-particle labels are bound.
 3. The method according to claim 1,wherein said fluidic force operates normally through a porous substrateon the surface of which said micro-particle labels are bound.
 4. Themethod according to claim 1, wherein the laminar fluid flow is pulsedfrom low or no flow to higher flows for a period or periods of time toeffect greater removal of non-specifically bound particles.
 5. Themethod according to claim 1, wherein said micro-particles comprisemicro-particles of size range of about 50 nm to about 10 μm.
 6. A methodfor controllably removing non-specifically bound micrometer-scaleparticle labels in binding assays, comprising: A. selecting a solidsubstrate; B. bonding a selected receptor onto said substrate; C.applying a ligand onto said substrate, whereby said ligand bondsspecifically with said select receptor; D. treating said substrate witha micrometer-scale particle label selected to bond specifically to oneof at least said ligand or to an intervening receptor that bindsspecifically to the ligand and to the micrometer- scale particle label;E. employing a fluidic force discrimination system to removenon-specifically bound micrometer-scale-particles labels, wherein saidfluidic force discrimination system comprises a laminar flow systemcharacterized by fluidic flow drag force which operates on saidmicrometer-scale particle labels for such a time and at such a flow rateuntil the ratio of specifically bound labels to non-specifically boundlabels increases to a desired degree, said micrometer-scale-particlesbeing detected by a detection means.
 7. The method according to claim 6,wherein said solid substrate is a bead array counter (BARC) microchipincorporating an array of magnetoresistive magnetic field sensors. 8.The method according to claim 6, wherein said substrate is a forcediscrimination assay sensor in the form of at least one of a solid orporous substrate of alumina, silicon, silica, or rigid polymer.
 9. Themethod according to claim 6, wherein receptor-ligand pairs are selectedfrom the group of antibody-antigen, enzyme-substrate, chelator-ion, orpolynucleic acid—complementary strand.
 10. The method according to claim6, wherein two or more receptors are bonded to said substrate, one ormore receptors being positive for and one or more receptors beingnegative for reaction towards a target ligand.
 11. The method accordingto claims 6, wherein said micrometer-scale labels are selected from thegroup consisting of magnetic, dielectric, radioactive, fluorescent,luminescent, or light scattering.
 12. The method according to claim 6,wherein said detection means is selected from the group of sensorsconsisting of magnetic, electric, nuclear-radiation, optical,electrochemical, thermal, and piezoelectric.
 13. A method foridentifying ligand-receptor pairs in binding assay measurement systems,comprising: A. selecting a substrate in a assay system onto which can bebonded a receptor molecule; B. reacting said substrate with saidreceptor molecule, wherein said molecule is bound to said substrate; C.reacting said substrate with a sample containing a ligand molecule,wherein said ligand molecule binds with said receptor molecule to form aligand-receptor pair, said ligand molecule being labeled with amicro-particle that can be detected; D. applying fluidic forcediscrimination method in a laminar flow system, wherein saidligand-receptor pairs are broken apart.
 14. The method according toclaim 13, wherein the flow rate applied by said fluidic force system tosaid substrate to break said ligand-receptor bonds is used to identifyvarious ligand-receptor bond pairs.
 15. A method for separating orsorting of populations of ligands by fluidics force discrimination,comprising A. selecting a substrate onto which can be bonded one or moretypes of receptor molecules; B. reacting said substrate with saidreceptor molecules, wherein said receptors are bound to said substrate;C. reacting said substrate with one or more complementary ligands,wherein said ligands bind with their said complementary receptormolecules to form ligand-receptor pairs, said ligand molecules beinglabeled with a micro-particle. D. applying the fluidic forcediscrimination method to said substrate, wherein the bonds of theweakest of said complementary ligand-receptor pairs are broken and thereleased ligands and beads can be separated by flow or other means fromthe substrate and ligands attached to the substrate.
 16. The methodaccording to claim 15, wherein the flow rate applied by said fluidicforce system is applied at a magnitude and for a time sufficient toremove a selected number of ligands of a desired type, preparing asubstrate with desired surface density of remaining ligands.
 17. Amethod for identifying molecular bonds in assay measurement systems,comprising: A. selecting a substrate onto which can be bonded one ormore types of receptor molecules; B. reacting said substrate with one ormore said molecules, wherein said molecules are bound to said substrate;said molecule being labeled with a micro-particle that can be detected;C. applying fluidic force discrimination method in a laminar flowsystem, wherein said molecular bonds are broken apart.
 18. The methodaccording to claim 17, wherein the flow rate applied by said fluidicforce system to said substrate to break said molecular bonds is used toidentify the bonds.
 19. A method for separating or sorting ofpopulations of molecules by fluidics force discrimination, comprising A.selecting a substrate onto which can be bonded one or more types ofreceptor molecules; B. reacting said substrate with said molecules,wherein said molecules are bound to said substrate; said molecules beinglabeled with a micro-particle. C. applying the fluidic forcediscrimination method to said substrate, wherein the bonds of theweakest of the molecular bonds are broken and the released molecules canbe separated by flow or other means from the substrate and moleculesattached to the substrate.
 20. The method according to claim 19, whereinthe flow rate applied by said fluidic force system is applied at amagnitude and for a time sufficient to remove a selected number ofsecondary binding members of desired types, preparing a substrate ofdesired surface density of remaining secondary binding members.
 21. Themethod of claim 1, further comprising flowing fluid through a flow cell,in cycles, for a predetermined time period followed by a zero flow ratefor a predetermined time period.
 22. The method of claim 21, wherein theflowing of fluid is controlled to rupture bonds between a plurality ofparticle-labeled targets and substrate-bound receptors.
 23. The methodof claim 21, wherein volumetric flow of fluid through the flow cell isprovided by Q=s³wP/12 l, where Q=volumetric flow of fluid; s, w, lrepresent height, width, length, respectively of flow cell;$\begin{matrix}{{P = {{pressure}\quad {differential}\quad {across}\quad {flow}\quad {cell}\quad {length}}};} \\{= {{viscosity}\quad {of}\quad {fluid}\quad {flowing}\quad {through}\quad {flow}\quad {{cell}.}}}\end{matrix}$


24. A method for removing non-specifically bound particle labels inbinding assays, comprising: treating a capture surface with a detectableparticle label bondable to a target molecule; employing a fluidic forcediscrimination system to remove non-specifically bound detectablelabels, wherein said fluidic force discrimination system includes alaminar flow system, the flow system being operated for a predeterminedtime period and at a predetermined flow rate to establish a higher ratioof specifically bound particles to non-specifically bound particles; andflowing fluid through the fluidic force discrimination system configuredto enable application of controlled, variable forces to disassociate thenon-specifically bound detectable labels from the capture surface. 25.The method of claim 24, wherein the fluidic force discrimination systemcomprises a flow cell, the flow cell being attached to the capturesurface to enable flowing of the fluid to disassociate thenon-specifically bound detectable labels.
 26. The method of claim 25,wherein volumetric flow of fluid through the flow cell is provided byQ=s³wP/12 l, where Q=volumetric flow of fluid; s, w, l represent height,width, length, respectively of flow cell; $\begin{matrix}{{P = {{pressure}\quad {differential}\quad {across}\quad {flow}\quad {cell}\quad {length}}};} \\{= {{viscosity}\quad {of}\quad {fluid}\quad {flowing}\quad {through}\quad {flow}\quad {{cell}.}}}\end{matrix}$


27. A method for removing non-specifically bound particle labels inbinding assays, comprising: treating a substrate having a capturesurface with a detectable particle label bondable to a target molecule;providing the substrate in a fluidic force discrimination system havinga fixed fluid volume; and moving the substrate within the fluid volumeto disassociate non-specifically bound detectable labels, the moving ofthe substrate being performed for a predetermined time period and at apredetermined rate to generate controlled, variable forces todisassociate the non-specifically bound detectable labels from thecapture surface thereby establishing a higher ratio of specificallybound particles to non-specifically bound particles.
 28. The method ofclaim 27, wherein the moving of the substrate is performed in cycles bymoving the substrate for a predetermined time period within the fluidvolume followed by stopping the movement of the substrate for apredetermined time period to generate laminar flow conditions havingforces to disassociate the non-specifically bound detectable labels.